The cranial bones protect the brain and are associated with pathologies ranging from congenital cranial bone anomalies to acquired traumatic or post-surgical skeletal defects. Thus, studying cranial bone development has clinical implications in addition to offering major mechanistic insights. Cranial bone progenitors, primitive cells destined to become the bone matrix-secreting osteoblasts, require specific cues from inter-cellular signaling pathways for fate selection, proliferation, and terminal differentiation. However, the temporal requirements and tissue origins of the signaling cues that drive cranial bone development remain obscure. The specific hypothesis to be tested is the following: Wnt signaling from the cranial surface ectoderm is required for the fate, proliferation, and differentiation of the cranial bones. The hypothesis is based on observations that 1) Wnt ligands are expressed in cranial surface ectoderm which gives rise to the craniofacial epidermis, 2) cranial bone progenitor cells organize beneath the cranial surface ectoderm layer and 3) genetic deletion of the central transducer of the Wnt signaling pathway in the cranial bone lineage results in loss of ossification of the cranial bones. This proposal will combine cell lineage analysis and conditional functional genetics in mice to address spatiotemporal requirements of the Wnt signaling pathway in cranial bone development. The approach will utilize novel conditional mutants to test the roles of Wnt signaling in cranial bone fate decisions and to query the surface ectoderm as the source of these cues. The Specific Aims are to: 1) Define the function of Wnt signaling in cranial bone development using a novel functional mutant that affects both the frontal and parietal bone plates. These studies will be the first to precisely define the requirement and sufficiency of Wnt signaling in cranial bone cell fate selection and ossification. 2) Define a novel role for surface ectoderm Wnts in cranial bone cell fate selection using a genetic tool that eliminates all Wnt ligand production in the surface ectoderm. Tissue-specific deletion of Wnt production will show if the surface ectoderm is the source of Wnts in cranial bone fate decisions. The results of this study will provide the first comprehensive view of the role of Wnt signaling in cranial bone fate selection and differentiation and test if the surface ectoderm is the source of the Wnt ligands. Public Health Relevance: These studies are congruent with the long term goal of defining the signaling cues and tissue-tissue context required for cranial bone fate decisions. Delineating the spatiotemporal signaling requirements of cranial bone development is a necessary prerequisite to understanding congenital cranial bone anomalies and developing cell-based therapies which recapitulate functional bone.